Polypeptide ligands for targeting cartilage and methods of use thereof

ABSTRACT

Ligands that specifically bind to articular cartilage tissues are disclosed, including uses for targeting therapeutics towards articular cartilage tissue and new materials for articular cartilage. The ligands are effective in vivo to target therapeutic materials to articular cartilage.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/545,819 filedOct. 10, 2006, which is hereby incorporated herein by reference.

TECHNICAL FIELD

The technical field of the invention is generally related to delivery oftherapeutic agents to cartilage tissues using polypeptides thatspecifically bind cartilage.

BACKGROUND

Cartilage lesions are common and can pose difficulties both in diagnosisand treatment. A lesion can either be a defect or a focal cartilagedegradation without visible disruption of the cartilage matrix. Suchlesions can result from an injury as in sports, disease, or aging. Theprognosis of an articular cartilage defect varies according to age,mechanism of injury, site, size, associated injuries and treatmentreceived.

SUMMARY OF THE INVENTION

The invention, however, provides treatments for cartilage injury. Someaspects of the inventions are substantially pure polypeptides comprisingan amino acid sequence of WYRGRL (SEQ ID NO:1), DPHFHL (SEQ ID NO:2), orRVMLVR (SEQ ID NO:3), or a conservative substitution thereof, or anucleic acid encoding the same. Such polypeptides specifically bindcartilage tissue. Such polypeptides may also include a therapeuticagent.

Some inventive methods are related to treating cartilage of a mammalcomprising administering to the mammal a pharmaceutically acceptablecomposition that comprises a nucleic acid encoding a polypeptide thatspecifically binds a cartilage tissue. Such polypeptide may also encodea therapeutic agent that, for example, treats a joint or tissue.

Other aspects of the invention relate to a delivery system fordelivering a therapeutic agent comprising: a substantially purifiedpreparation that comprises a pharmaceutically acceptable excipient, atherapeutic agent, and a polypeptide ligand comprising an amino acidsequence in the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, and conservative substitutions thereto. The polypeptide ligandsmay specifically bind to cartilage tissue for targeted delivery of thetherapeutic agent to cartilage tissues. The therapeutic agent maycomprise, for example, a drug, a visualization agent, or a therapeuticpolypeptide. The delivery system may include, for example, a collectionof nanoparticles having an average diameter of between about 10 nm andabout 200 nm, wherein the nanoparticles comprise the therapeutic agentand the polypeptide ligand.

Other embodiments relate to a biomaterial comprising a polymer and asubstantially pure polypeptide comprising an amino acid sequence of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a conservative substitutionthereof, wherein the polypeptide specifically binds to cartilage tissueand the polymer is free of amino acids and has a molecular weight of atleast 400. A variant of WYRGRL (SEQ ID NO:1) is WYRGRLC (SEQ ID NO:4),with the C-terminal residue being used as a chemical linker.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 a is a bar graph that shows amplification of three phage clonesidentified by biopanning and demonstrates that they all grow at equalrates comparable to the random library. Final selection of C1-3 wastherefore not affected by differences in growth during amplification.

FIG. 1 b is a bar graph that demonstrates the competitive binding assayin which only phage clone C1-3 and C1-C1 could be recovered.

FIG. 1 c is a bar graph that shows binding specificity of C1-3 and C1-C1to articular cartilage. Binding of these phage clones to synovialmembrane resulted in a lower phage recovery by two orders of magnitude,reflecting non-specific binding. Error bars indicate mean±standarddeviation from three independent experiments.

FIG. 2 is a graph showing inhibition of cartilage binding of C1-3 by thesynthetic polypeptide WYRGRLC (SEQ ID NO:4). Data represents thepercentage of maximal phage binding of clone C1-3 obtained in theabsence of synthetic polypeptide. Error bars indicate mean±standarddeviation from three independent experiments.

FIG. 3 is a bar graph showing inhibition of cartilage binding of C1-3phage by 10₁ μM of synthetic polypeptide vs. WYRGRLC (SEQ ID NO:4)—PPSnanoparticles and 10 μM of synthetic mismatch polypeptide. WYRGRLC (SEQID NO:4)—PPS nanoparticles exhibit similar binding than the syntheticpolypeptide, whereas the synthetic mismatch polypeptide does not resultin a significant decrease of phage titer. Error bars indicatemean±standard deviation from three independent experiments.

FIG. 4 is a bar graph showing relative accumulation in articularcartilage in vivo of nanoparticles decorated with WYRGRLC (SEQ ID NO:4)compared to control nanoparticles. Error bars indicate mean±standarddeviation from three independent experiments.

DETAILED DESCRIPTION Introduction

Ligands that specifically bind to articular cartilage tissues have beendiscovered.

These articular cartilage tissue-binding ligands have given rise to newtechniques to target therapeutics towards articular cartilage tissue andnew materials for treatment of articular cartilage defects. The ligandsare effective in vivo to target therapeutic materials to articularcartilage.

Three of the cartilage tissue-binding ligands are polypeptides with theamino acid sequence of WYRGRL (SEQ ID NO:1), DPHFHL (SEQ ID NO:2), orRVMLVR (SEQ ID NO:3). Other ligands are polypeptides with sequences thathave conservative substitutions of one of SEQ ID NOs. 1, 2, or 3. Ligandis a term that refers to a chemical moiety that has specific binding toa target molecule. A target refers to a predetermined molecule, tissue,or location that the user intends to bind with the ligand. Thus targeteddelivery to a tissue refers to delivering a molecule to the intendedtarget tissue; a therapeutic agent delivered to a target may be intendedto act on the target itself or on some other molecule or cell, e.g., acell or tissue that is near the target.

One application of the cartilage tissue-binding ligands is for targetingtherapeutic agents to articular cartilage, commonly referred to asintra-articular drug delivery when drugs are delivered. Intra-articulardrug delivery has been termed a major challenge due to the shortresidence times of intra-articular injected drugs. Drugs injected bythemselves tend to diffuse away rapidly or be otherwise rapidly taken upinto the circulation, thus causing low bioavailability of the drug atthe cartilage and unwanted systemic effects. Despite sustained-releasedrugs, only a few reports exist on the intra-articular use of anysustained-release formulations²⁰⁻²⁶. Mostly albumin andpoly(lactic-co-glycolic acid) (PLGA) have been used as biocompatible andbiodegradable polymers for this purpose either in the form of gels²² oras microspheres²³⁻²⁶. Such approaches rely, however, on degradation overtime to achieve sustained-release within the joint, a strategy that islimited by the properties of available biomaterials, and which makes thedrugs available only when the material degrades. Targeting the deliverysystem to articular cartilage with tissue-binding ligands makes thetissue itself a reservoir for drug release to the site of the diseaseprocess as opposed to release in the joint cavity.

The cartilage tissue-binding ligands are also useful to bind cartilagetissue in vitro for diagnostic, assay, or imaging purposes. Forinstance, sections of tissue may be exposed to cartilage tissue-bindingligands that are also bound to a fluorescent molecule or other imagingagent to visualize the location of the cartilage tissue. Or, forinstance, the cartilage tissue-binding ligands may be used in affinitychromatography to isolate the tissue.

In some aspects, therefore, articular cartilage tissue-binding ligandsdescribed herein enable new techniques for controlled release byenabling sustained localization through specific binding to cartilage,and their accompanying formulations increase intra-articularbioavailability of delivered drugs or other therapeutic agents, which isbeneficial for various disease processes, e.g., those involving thesynovium such as rheumatoid arthritis or inflammation in clinicallymanifest osteoarthritis. Because convective transport of solutes intocartilage is impaired due to the inherent properties of thistissue^(27,28), the bioavailability of drugs in the cartilage matrix,which is the primary site of the disease process in osteoarthritis, canbe enhanced by sustained release systems that reside in the matrixitself. Targeting of the cartilage matrix as described herein istherefore useful for general intra-articular therapeutic agent delivery,such as targeting of the cartilage matrix for delivery of therapeuticagents to treat cartilage degradation in osteoarthritis and protectcartilage in conditions like rheumatoid arthritis, bacterial andreactive arthritis.

The cartilage tissue-binding ligands are thus useful to directtherapeutic agents to a target. A therapeutic agent refers to a moleculefor delivery to a target to accomplish a desirable medical or scientificfunction, and the term includes drugs and imaging agents. Examples oftherapeutic agents are matrix metalloproteinase (MMP) inhibitors,aggrecanase inhibitors, COX-inhibitors and other non-steroidalanti-inflammatory drugs (NSAIDs), glucosamin, diacerhein, methotrexate,steroids, immunosuppressing drugs (rapamycin, cyclosporine), proteintherapeutics (growth factors, tissue inhibitors of matrixmetalloproteinases), and oligonucleotides (e.g., siRNA, shRNA, miRNA).Examples of imaging agents are fluorescent markers, radio-opaquematerials, magnetic resonance imaging contrast agents, x-ray imagingagents, radiopharmaceutical imaging agents, ultrasound imaging agents,and optical imaging agents.

Ligand Discovery and Experimental Data

The development of phage display of random peptides on the minor coatproteins (pIII) of bacteriophages has allowed use of affinitypurification, in a process called biopanning⁵, to identify specificpeptides for precise targeting of multiple tissues, both in vitro⁶ andin vivo in animals and even humans⁷⁻⁹. As an example, Arap et al. havesucceeded in mapping the human vasculature and specifically targetingthe microvasculature of adipose tissue in mice. Another study identifieda synovium-specific homing peptide by phage display. Human synovialgrafts were transplanted into SCID mice and biopanning was carried outin vivo which resulted in the identification of phages with homingpeptides specific for the microvascular endothelium of synovialtissue¹¹.

In order to identify peptide sequences which bind to the articularcartilage extra-cellular matrix, it should first be appreciated that anin vivo approach to biopanning must address the challenges presented bythe dense organization of extra-cellular matrix molecules. Indeed, thecollagen II fiber network of articular cartilage has a reported meshsize of 60 nm in the superficial zone¹² and largest gaps between theside chains of proteoglycan aggregates have been described to be as lowas 20 nm¹³. Biopanning was therefore carried out not in vivo, butinstead was adapted for use with ex vivo materials. Specifically, slicedbovine cartilage was used, wherein the extra-cellular matrix was exposedfor affinity purification of binding phage virions.

Materials The phage display library fUSE5/6-mer based on filamentousphage strain fd-tet was received from the University of Missouri,Columbia. Cartilage grafts, synovial fluid and synovial membrane wereharvested from bovine shoulders obtained from the local slaughterhouse.Cartilage grafts were stored in 0.1% sodium azide and proteaseinhibitors at 4° C. and used within 72 hours. Buffers and solutions usedfor phage-display screening: Blocking solution (0.1M NaHCO₃, 1% BSA, pH8.5), wash buffer (PBS, Tween 20 0.1-1%), elution solution (50 mMglycine-HCl, pH 2.0), neutralisation buffer (0.2M NaHPO₄). Solvents andreagents for nanoparticle synthesis were purchased from Sigma-Aldrich(Buchs, Switzerland). All peptides (synthesis chemicals fromNovabiochem, Lautelfingen, Switzerland) were synthesized on solid resinusing an automated peptide synthesizer (CHEMSPEED PSW 1100, Augst,Switzerland) with standard F-moc chemistry. Purification was performedon a Waters ultrapurification system using a Waters ATLANTIS dC₁₈semi-preparative column and peptides collected according to theirmolecular mass analyzed by time-of-flight (TOF) mass spectrometry.Screening of phage-displayed combinatorial peptide library and bindingassays Peptides for binding to the articular cartilage matrix wereselected by exposing a fUSE5/6-mer library to bovine cartilage grafts,which provided 6.4×10⁷ different phage clones with 6-amino acidlinear-peptide inserts displayed on the minor coat protein offilamentous phage^(5,18). Cartilage grafts were harvested with 8 and 4mm biopsy cutters (two-sided surface 1 cm² and 0.25 cm²). A slice withthe intact articular surface was removed to expose the phages only tothe cartilage matrix below the surface. Affinity selection was preformedin polystyrene 48-well plates, which were blocked for nonspecificadhesion with a blocking solution containing 1% BSA for 2 hours prior toscreening. A total of 5 screening rounds was carried out. In the firstround 10¹³ and in subsequent rounds 10¹² phage virions per ml wereexposed to the cartilage graft, washed with PBS/Tween 20 and eluted atlow pH. While the first round was supposed to give a high yield at lowstringency, subsequent rounds were carried out with increasingstringency to select stronger binders. Conditions were increased from 4hours of binding at RT and washing with PBS/0.1% Tween to 30 minutesbinding at 37° C. with 220 RPM, washing with PBS/1% Tween 20 and thecartilage surface decreased from 1 cm² to 0.25 cm² in round five. Elutedphage was amplified overnight in E. coli strain TG1 in 2×YT medium andpurified by two times PEG/NaCl (2M, 25%) precipitation. In the firstround of screening, negative screens against the intact articularsurface as well as synovial fluid (50% diluted in PBS to lowerviscosity) were carried out in order to eliminate phages binding tothese targets. Quantitative titer counts were obtained by spot titeringof 15 μl of phage/bacterial culture onto LB-agar/tetracyclin plates andare given in transducing units (TU)/ml. The polypeptide sequences ofaffinity selected phage specific to the articular cartilage matrix wasdetermined by DNA sequencing (Microsynth AG, Balgach, Switzerland) usingthe sequencing primer 5′-CAT GTA CCG TAA CAC TGA G (SEQ ID NO:5).Binding specificity was determined by exposing selected phage clones(10⁸ TU/ml) to articular cartilage (0.25 cm²) with and without synovialfluid, to synovial membrane (3×4 mm, 0.24 cm²) and to polystyrene afterblocking. Titer counts were obtained by spot titering. Competetivebinding was probed by exposing a mix of selected phage clones andfUSE5/6-mer library (each 10⁸ TU/ml) in the presence of synovial fluidto articular cartilage for 30 min. at 37° C., 220 RPM and washed withPBS/1% Tween 20. The phage clones were identified by DNA sequencing andthe corresponding titer counts calculated. For competitive bindingagainst free polypeptide or nanoparticles and to obtain a dose-responsecurve, 10⁸ TU/ml of phage and its free polypeptide in differentconcentrations or nanoparticles were mixed, exposed to articularcartilage and titer counts determined. All experiments have been carriedout in triplicate and repeated for confirmation.Nanoparticle Synthesis Poly(propylene sulfide) nanoparticles wereprepared as described elsewhere¹⁴. Briefly, for nanoparticles between 30and 40 nm, a monomer emulsion is prepared by dissolving 1.6% (w/v) ofPluronic F-127 (MW 12600) in 10 ml of degassed, double-distilled andfiltered water. The system is continuously stirred and purged with argonfor 60-90 min. Propylene sulfide is added at a Pluronics/monomer ratioof 0.4 (w/w). The initiator, pentaerythritol tetrathioester (TTE)(synthesized as described previously¹⁴), is deprotected by mixing with amolar equivalent of 0.5M sodium methanoate and stirred under argon for 5minutes. The deprotected initiator is then added to the emulsion. Fiveminutes later, 60 μl of diaza[5.4.0] bicycloundec-7-ene (DBU) is addedand the reaction stirred under inert conditions for 6 hours. Exposure toair yields disulfide crosslinking of the particle core. Particles aresubsequently purified from remaining monomers and base by 2 days ofrepeated dialysis against ultrapure water through a membrane with a MWCOof 6-8 kDa (Spectra/Por). Free Pluronic F-127 is removed in a seconddialysis step through 300 kDa membranes. Particle size is measured afterdialysis by dynamic light scattering (ZETASIZER NANO ZS, MalvernInstruments, Malvern, UK). For preparation of surface-functionalizednanoparticles, polypeptides are conjugated to Pluronics-F127 prior tonanoparticle synthesis. In order to derivatize Pluronics F-127 withvinyl sulfone, 400 ml of toluene and 15 g of Pluronic F-127 wereintroduced in a 3-neck round bottom flask connected to a Soxhlett filledwith glass wool and dry molecular sieves and a cooling tube. Pluronicwas dried azeotropically during 4 h. The dried Pluronic in toluene wascooled in an ice-bath and sodium hydride was added in a 5 equimolarexcess compared with Pluronic-OH-groups. The reaction was stirred for 15minutes and divinyl sulfone was added in a 15 molar excess and thereaction was carried out in the dark for 5 days at RT under argon. Thereaction solution was filtrated through a celite filter cake,concentrated by rotary evaporation and then precipitated 5 times inice-cold diethylether. The polymer was dried under vacuum and storedunder argon at −20° C. Derivatization was confirmed with ¹H-NMR(CDCl₃):=1.1 (m, PPG CH₃), 3.4 (m, PPG CH), 3.5 (m, PPG CH₂), 3.65 (m,PEG CH₂), 6.1 and 6.4 (d, 1H each, CH₂═CH—SO₂—), 6.85 (q, ¹H,CH₂═CH—SO₂—) ppm. A degree of end group derivatization of 88% wasdetermined by ¹H-NMR. All polypeptides were acetylated at the N-terminusto prevent reaction with the α-amine and synthesized with a cysteine atthe C-terminus to be conjugated to Pluronics-di-vinyl sulfone byMichael-type addition via the free thiol²⁹. For conjugation, 1.6 mMPluronics-di-vinyl sulfone is stirred in triethanolamine buffer at pH8.5 until it is completely dissolved and 2 mM of polypeptide is addedand stirred for 3 hours at RT. Conjugation is confirmed by absent vinylsulfone peaks by ¹H-NMR in methanol. Nanoparticles are prepared asdescribed above with 10% surface functionalisation corresponding to afraction of 0.16% (w/v) of conjugated Pluronic. The presence of thepolypeptide on the particle is confirmed by measuring the zeta potentialby dynamic light scattering (ZETASIZER NANO ZS, Malvern Instruments,Malvern, UK) after particle synthesis. The nanoparticles werefluorescently labelled with 6-iodoacetamide fluorescein (6-IAF,Molecular Probes, Eugene, Oreg.) or Alexa FLUOR 488 maleimide by addinglabel at 1 mg/ml of nanoparticle solution in 10 mM Tris-HCl pH 8.5 andstirred in the dark for 2 h at room temperature. Unreacted label wasquenched by adding 5 mg L-Cysteine. Purification was accomplished bydialysis for 24 hours against a membrane with MWCO of 24 kDa in 5 mM PBSwith two buffer shifts.In vivo injection Labeled nanoparticles were injected into knee jointsof 4-6 weeks old C57BL/6 mice at a concentration of 1% (w/v) in a volumeof 5 μl using a 25 μl Hamilton syringe with Cheney reproducibilityadapter and 30 G needles (animal experimentation protocol approved bythe local review board, authorisation no. 1894). The mice wereanesthetized with isoflurane. The animals were euthanized byCO₂-asphyxiation after 24 hours. The knee joints were harvested andfreed from surrounding muscle by microdissection.Confocal microscopy and image analysis The sections were analyzed byconfocal laser scanning microscopy using a Zeiss LSM510 meta.Fluorescence was extracted by emission fingerprinting to reduceautofluorescence of cartilage tissues. The pinhole was adjusted usingTETRASPECK fluorescent microspheres (Invitrogen, T14792, Carlsbad,Calif.). Images for analysis were obtained using a Zeiss 63× APOCHROMATobjective in 10 different locations per joint with a z-stack of 10images each. Image deconvolution was accomplished by Huygens software.Image analysis was done by ImageJ.

Affinity Selection of Phage Display Library and Binding Assay

Cartilage grafts were incubated with the fUSES peptide on phage displaylibrary, which expressed linear 6-mer random peptides on minor coatproteins (pIII) with a diversity of 6.4×10⁷. The sequences correspondingto the polypeptides displayed on the phage virions were determined byDNA sequencing after rounds 3 and 5 of affinity selection (panning).After round 3, sequencing did not reveal a consensus motif in theselected polypeptides. Sequencing of round 5 yielded three differentphage clones C1-3 (having SEQ ID NO:1), C1-C1 (having SEQ ID NO:2) andC1-F1 (having SEQ ID NO:3), whereas C1-3 appeared in 94 out of 96sequenced clones and C1-C1 and C1-F1 both only appeared once. To ensurethat the selection of the three phage clones was not the result ofdifferences in their amplification rates compared to other phages,overnight amplification of 10⁶ particles/ml of the three selected clonesand the random fUSE5 phage library was performed in a bacterial culture.As shown in FIG. 1 a, all amplification rates are equal to the rate ofthe random library. This suggests that the three phage clones were notselected in the panning process because of differences in theiramplification speed, which would have biased the library in their favor.A competetive binding assay was carried out to assess the relativebinding strength of the selected phage clones against each other. Equalamounts of the three phage clones and the random fUSES library weremixed and exposed to cartilage grafts (0.25 cm²). The correspondingtiter counts of recovered phage virions were determined by DNAsequencing of 96 colonies in triplicate. It is demonstrated in FIG. 1 bthat only C1-3 and C1-C1 have been recovered, thereby indicating theirsuperior binding strength over C1-F1 and the random library. Inaddition, C1-3 has a higher titer count of almost one order of magnitudethan C1-C1 which further demonstrates its dominant binding which wasalready suggested by its frequent appearance after round 5.

The binding specificity of C1-3 and C1-C1 to articular cartilage wasevaluated by exposing the phage clones to articular cartilage (0.25 cm²)and synovial membrane (0.24 cm²) and comparing to random binding of thefUSES phage library. Furthermore, the effect of the presence of synovialfluid on phage binding was probed by adding an equal volume of bovinesynovial fluid to the phages, which dilutes the synovial fluid by afactor of 2. FIG. 1 c shows that both C1-3 and C1-C1 exhibit specificbinding to articular cartilage over synovial membrane by two orders ofmagnitude and that the addition of synovial fluid does not yield asignificant drop in phage binding to articular cartilage. Binding of thespecific phage clones to synovial membrane seems to reflect backgroundphage binding as the random phage library fUSES bound to the synovialmembrane to the same extent. In addition, phage titers of fUSE5 toarticular cartilage were at the same level as to synovial membrane,further indicating specific binding of C1-3 and C1-C1. Phage was exposedto polystyrene wells after blocking them with BSA because it iscontained in the plasticware in which affinity selection was carriedout. No background phage binding to polystyrene was detectable, however.

Affinity selection of fUSE5/6-mer phage display library resulted in thediscovery the phage clone C1-3 (polypeptide WYRGRL, SEQ ID NO:1) whichexhibits both binding specificity to articular cartilage and dominantbinding compared to other phage clones. Specific binding in the contextof a polypeptide refers to the binding of the polypeptide specificallyto the target of interest as opposed to other molecules.

Competetive Binding of C1-3 Against Free Polypeptides WYRGRLC andYRLGRWC

Based on affinity selection and the binding assays reported herein, the6-mer polypeptide insert WYRGRL (SEQ ID NO:1) of clone C1-3 as well asits scrambled mismatch YRLGRW (SEQ ID NO:6) were synthesized on solidresin using standard Fmoc chemistry. The N-terminal amino acids areacetylated and a cysteine was added to the C-terminus of thepolypeptides for bioconjugation to vinyl sulfone by Michael-typeaddition via the free thiol. In order to further characterize thebinding properties of WYRGRLC (SEQ ID NO:4) to articular cartilage, acompetetive binding assay against the phage clone C1-3 was performed byexposing the C1-3 and the WYRGRLC (SEQ ID NO:4) to the cartilage. Adose-response curve was determined by serial dilutions of thepolypeptide ranging from 50 nM to 10 μM and mixing them with 10⁸ TU/mlof phage. The titer counts of phage recovered gradually decreased by twoorders of magnitude as the concentration of the free polypeptide insolution was increased (FIG. 2). An IC50 of about 200 nM can beestimated from the curve in FIG. 2.

Conjugation of Polypeptide to Pluronic F-127 and Nanoparticle Synthesis

In order to functionalize poly(propylene sulfide) (PPS) nanoparticles,Pluronic F-127 was derivatized with vinyl sulfone. The polypeptides wereconjugated to Pluronic-di-vinyl sulfone via the free thiol in theC-terminal cysteine by Michael-type addition.

Conjugation was confirmed by ¹H-NMR in methanol by the absence of peaksspecific to vinyl sulfone. PPS nanoparticles were then prepared byinverse emulsion polymerization with Pluronic F-127 (90%) andpolypeptide-conjugated Pluronic F-127 (10%) serving as the emulsifier.Because Pluronic as the emulsifier remains on the particle surface, theconjugated polypeptide is displayed on the surface of the nanoparticles,thereby adding the targeting functionality of the polypeptide to theparticles. Size measurements by dynamic light scattering (ZETASIZER NANOZS, Malvern Instruments, Malvern, UK) revealed a size by volume of 38 nmfor PPS particles displaying WYRGRLC (SEQ ID NO:4) (polydispersity indexPDI 0.221), 31 nm for particles displaying YRLGRC (SEQ ID NO:7) PDI0.412) and 37 nm for non-conjugated PPS particles (PDI 0.212). Thezeta-potential of non-conjugated PPS nanoparticles is about neutral(−2.64±8.97 mV).

Due to the positive charges of the polypeptide, the zeta-potential ofconjugated nanoparticles shifted to +17.8±3.45 mV, which furtherconfirms the presence of the polypeptide on the nanoparticle surface.

WYRGRLC (SEQ ID NO:4)—PPS nanoparticles at 2% (w/v) were subjected to acompetetive binding assay against the free polypeptides WYRGRLC (SEQ IDNO:4) and YRLGRC (SEQ ID NO:7) at concentrations of 10 μM each.Accordingly, phage clone C1-3 was exposed to the cartilage in thepresence of WYRGRLC (SEQ ID NO:4)—PPS nanoparticles, WYRGRLC (SEQ IDNO:4) or YRLGRC (SEQ ID NO:7), and the amount of C1-3 phage binding tothe cartilage relative to a control C1-3 phage without competitiveinhibitors was measured. The results in FIG. 3 show that WYRGRLC (SEQ IDNO:4)—PPS nanoparticles exhibit similar binding as the correspondingfree polypeptide (WYRGRLC (SEQ ID NO:4)—PPS10.4±6% and WYRGRLC (SEQ IDNO:4)13.9±2.8% of control), whereas the YRLGRC (SEQ ID NO:7) did notbind competetively and thus did not result in a significant drop ofphage titer (92±12% of control). Conjugated PPS nanoparticles at adegree of surface functionalisation of 10% at 2% (w/v) therefore seem tohave similar binding to articular cartilage as the free polypeptideWYRGRLC (SEQ ID NO:4) at 10 μM.

Active Targeting of Articular Cartilage In Vivo

WYRGRLC (SEQ ID NO:4)—PPS and PPS nanoparticles were labeled with 6-IAFand dialysed for 2 days with at least 2 buffer shifts to ensure that nofree label is still in the solution. A volume of 5 μl of thenanoparticles was injected into the knee joints of 4-6 weeks old C57BL/6mice. Three mice were injected with WYRGRLC (SEQ ID NO:4)—PPS in theright and PPS particles in the left knee joint. In order to doreproducible injections a 250 Hamilton syringe (Hamilton Europe,Bonaduz, Switzerland) with a Cheney reproducibility adapter was used forantero-lateral parapatellar injection with a 30 G needle. Cryosectionswhich were obtained after 24 hrs were analysed by confocal laserscanning microscopy. Quantification of fluorescent dots per cartilagevolume as determined by sampling of z-stacks with 10 planes in 10different locations per joint revelead an increase in particleaccumulation from 29.0±1.5% for PPS particles to 83.8±4.0% for WYRGRLC(SEQ ID NO:4)—PPS particles (FIG. 4). While there is an obviousfavorable accumulation of functionalized nanoparticles in the articularcartilage matrix after 24 hours, nanoparticles accumulate in the wholejoint at a concentration of 2% (w/v) and enter meniscal and ligamentoustissues in addition to the synovial membrane.

Discussion

Several methods exist for affinity selection of binding proteins orpolypeptides such as phage display^(s), yeast surface display¹⁵, mRNAdisplay¹⁶ or peptide-on-bead display¹⁷. Herein, phage display using thefUSE5/6-mer library based on the filamentous phage vector fd-tet¹⁸ wasused to select short peptides which bind to the articular cartilagematrix. Embodiments of the invention include using affinity selection ofbinding proteins or peptides against cartilage ex vivo.

In this case, biopanning was carried out against slices of bovinecartilage. Conditions in the binding step were chosen with increasingstringency from round 1 to 5 in order to favor binding of high affinitypolypeptides. The selected sequences obtained from DNA sequencing of 96clones have been evaluated in a competetive binding assay.

Two sequences C1-3 and C1-C1 exhibited stronger competetive binding thanC1-F1 and random phages from the fUSE5 library (FIG. 1 b). Therefore,specificity of binding for C1-3 and C1-C1 to cartilage was furtherassessed. The phage clones were subjected to physiological conditions(37° C. and shaking) and binding specificity probed for cartilage,cartilage in the presence of synovial fluid and the synovial membrane.Phage titers of both C1-3 and C1-C1 were higher for cartilage than forsynovial membrane by two orders of magnitude. More importantly, bindingto the cartilage target was not impaired by the addition of synovialfluid (FIG. 1 c). This is likely to be the result of the negativescreening which was carried out during the first round of biopanning. Inorder to eliminate phages with polypeptide sequences that potentiallybind to constituents of the synovial fluid, the first screening wascarried out in the presence of synovial fluid, which was discardedincluding the phages before the binding phages were eluted off thecartilage slice. In order assess the relative binding affinity of thepolypeptide sequence, the corresponding polypeptide WYRGRLC (SEQ IDNO:4) was synthesized. In a competetive binding assay against the phageC1-3 displaying WYRGRL (SEQ ID NO:1) (10⁸ TU/ml) an IC₅₀ in the highnanomolar range of about 200 nM can be demonstrated (FIG. 2).

Binding specificity was conferred by the polypeptide sequence ratherthan the net positive charge of the polypeptide which sticksnon-specifically to negatively charged proteoglycans. This isdemonstrated in FIG. 3 in that 10 μM of WYRGRLC (SEQ ID NO:4) resultedin a decrease of phage titers close to 100fold. By contrast, 10 μM ofYRLGRC (SEQ ID NO:7), which comprises the same amino acids just inscrambled order, did not result in a significant decrease in phage titeras compared to control phage titers without polypeptide. Thus WYRGRLC(SEQ ID NO:4) is a short polypeptide with specific binding to articularcartilage which shows a dose dependent decrease in competetive bindingagainst phage C1-3. The other polypeptides, DPHFHL (SEQ ID NO:2) andRVMLVR (SEQ ID NO:3), which were discovered using the same experimentalmethods used for WYRGRL, could also be shown to have specific bindingusing these same techniques.

The polypeptides were synthesized such that they contain a cysteine atthe C-terminus. The free thiol of cysteine is used for bioconjugation byMichael-type addition to Pluronic-di-vinyl sulfone which serves as theemulsifier in nanoparticle synthesis and therefore remains displayed onthe particle surface. While this is a straightforward scheme for surfacefunctionalisation of nanoparticles synthesized by inverse emulsionpolymerization, conjugation of Pluronic requires excess of polypeptidedespite the favorable kinetics of Michael-type addition of free thiolsto vinyl sulfone, if conjugation close to 100% is to be achieved asevidenced by ¹H-NMR. In addition, some Pluronic is always lost duringnanoparticle synthesis. Alternative synthetic schemes therefore addsurface functionality to already-synthesized nanoparticles in order tolimit the amount of polypeptide needed.

Presence of the polypeptide on the nanoparticle surface as indicated bya shift in zeta-potential from neutral to positive was confirmed bycompetitive binding. WYRGRLC(SEQ ID NO:4)—PPS nanoparticles with adegree of surface functionalisation of 10% (w/w of total Pluronic used)at a concentration of 2% (w/v) against phage clone C1-3 (10⁸ TU/ml) toarticular cartilage resulted in a similar decrease of phage titers as 10μM of free polypeptide WYRGRLC (SEQ ID NO:4, FIG. 3).

Targeting the extra-cellular matrix of articular cartilage essentiallydepends on the ability of the drug delivery system to enter thecartilage matrix and to stay there. In passive targeting, thedistribution of nanoparticles in the joint is mainly governed by thecapability of tissue penetration and cellular uptake. While largerparticles do not enter, smaller ones are able to penetrate and reside inthe cartilage ECM. It is demonstrated in herein that nanoparticles (inthis case, nanoparticles with a mean volume diameter of 36 and 38 nm)are able to enter the articular cartilage ECM and meniscal tissue inaddition to the synovium. It is consistent with the literature thatsmall particles possess the ability to enter the cartilage ECM. It hasbeen demonstrated that adeno-associated viruses (AAV) with a meandiameter of 20-25 nm enter the articular cartilage matrix up to a depthof penetration of 450 μm in normal and 720 μm in degraded cartilage¹⁹.While both surface-functionalised and non-functionalised PPSnanoparticles enter the articular cartilage matrix, there is a markedincrease in the accumulation of WYRGRLC (SEQ ID NO:4)—PPS nanoparticlesover non-functionalised and therefore non-targeted PPS nanoparticles 24hours after intra-articular injection into the knee joint of mice (FIG.4). WYRGRLC(SEQ ID NO:4)—PPS nanoparticles therefore exhibit specifictargeting capability for the articular cartilage matrix. Although thebasic research has been performed with bovine cartilage, the polypeptidesequences described herein are expected to bind to human articularcartilage due to the general homology of these tissues. Bovine cartilageis an accepted model in cartilage research due to the very limitedavailability of healthy human cartilage.

Polypeptide Ligands Specific for Articular Cartilage

Three of the cartilage tissue-binding ligands are polypeptides with theamino acid sequence of WYRGRL (SEQ ID NO:1), DPHFHL (SEQ ID NO:2), orRVMLVR (SEQ ID NO:3). Other ligands are polypeptides or functionalpolypeptides with sequences that have conservative substitutions of oneof SEQ ID NOs. 1, 2, or 3.

Certain embodiments are directed to the subset of polypeptides that havea certain percentage identity to the disclosed sequences, or a certaindegree of substitution, with the subset being primarily, or only,functional polypeptides.

The binding activity of a polypeptide to cartilage may be determinedsimply by following experimental protocols as described herein. Forinstance, a polypeptide variant of one of the polypeptide ligands may belabeled with a marker (e.g., radioactive or fluorescent) and exposed tobovine cartilage to determine its binding affinity using well-knownprocedures. A binding assay may be performed using a simple fluorescencereadout using a plate reader by labeling polypeptide variants with afluorescent marker, e.g., 6-fluorescein iodoacetamide which reacts withthe free thiol of the cysteine. Using such a method, the bindingstrengths of polypeptide variants relative to e.g., WYRGRLC (SEQ IDNO:4) under given physiological conditions can be determined, e.g.,sequences made using conservative substitutions, truncations of thesequences to 5 or less amino acids, addition of flanking groups, orchanges or additions for adjusting sequences for solubility in aqueoussolution.

Polypeptides of various lengths may be used as appropriate for theparticular application. In general, polypeptides that contain thepolypeptide ligand sequences will exhibit specific binding if thepolypeptide is available for interaction with cartilage in vivo. Proteinfolding can affect the bioavailability of the polypeptide ligands.Accordingly, certain embodiments are directed to polypeptides that havea polypeptide ligand but do not occur in nature, and certain otherembodiments are directed to polypeptides having particular lengths,e.g., from 6 to 3000 residues, or 6-1000, or 6-100, or 6-50; artisanswill immediately appreciate that every value and range within theexplicitly articulated limits is contemplated. Moreover, the lowerlimits may be 4 or 5 instead of 6.

While polypeptides of 6 residues were extensively, tested, variants thathave 3, 4, or 5, residues can also be active and exhibit specificbinding, as well as conservative substitutions thereof. Accordingly,every contiguous 3, 4, and 5 residues in each sequence can be rapidlyscreened and tested for binding using the methods set forth herein,e.g., using the sequencing and binding assays, or with competitiveinhibition. Thus, in the case of SEQ ID NO:1, with W being the firstresidue and L the sixth residue, binding activity may be expected in thethree residues 1-3, 2-4, 3-5, and 4-6; for four residues, bindingactivity may be expected in 1-4, 2-5, and 3-6; for five residues,binding activity may be expected in positions 1-5 or 2-6. The ordinaryartisan, after reading this disclosure, will be able to quickly assaythis limited number of sequences. While some decrease in bindingactivity might be observed when the 6-residue sequences are truncated, acore group is expected to exhibit substantial binding. This expectationis based on general observations made with binding moieties in thesearts. For example, in phage display experiments, the peptide sequencesoften exhibit a consensus motif which usually does not involve allresidues displayed on the phage virion, e.g., in Arap et al. (NatureMedicine 2002; 8:121). Less stringent conditions or sequencing ofprevious rounds are more likely to give a consensus motif in differentpeptide sequences than one very strong binding sequence.

Certain embodiments provide various polypeptide sequences and/orpurified polypeptides. A polypeptide refers to a chain of amino acidresidues, regardless of post-translational modification (e.g.,phosphorylation or glycosylation) and/or complexation with additionalpolypeptides, synthesis into multisubunit complexes, with nucleic acidsand/or carbohydrates, or other molecules. Proteoglycans therefore alsoare referred to herein as polypeptides. As used herein, a “functionalpolypeptide” is a polypeptide that is capable of promoting the indicatedfunction. Polypeptides can be produced by a number of methods, many ofwhich are well known in the art. For example, polypeptides can beobtained by extraction from a natural source (e.g., from isolated cells,tissues or bodily fluids), by expression of a recombinant nucleic acidencoding the polypeptide, or by chemical synthesis. Polypeptides can beproduced by, for example, recombinant technology, and expression vectorsencoding the polypeptide introduced into host cells (e.g., bytransformation or transfection) for expression of the encodedpolypeptide.

There are a variety of conservative changes that can generally be madeto an amino acid sequence without altering activity. These changes aretermed conservative substitutions or mutations; that is, an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic can be substituted for another amino acid. Substitutesfor an amino acid sequence may be selected from other members of theclass to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and tyrosine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Such alterationsare not expected to substantially affect apparent molecular weight asdetermined by polyacrylamide gel electrophoresis or isoelectric point.Exemplary conservative substitutions include, but are not limited to,Lys for Arg and vice versa to maintain a positive charge; Glu for Aspand vice versa to maintain a negative charge; Ser for Thr so that a free—OH is maintained; and Gln for Asn to maintain a free NH₂. Moreover,point mutations, deletions, and insertions of the polypeptide sequencesor corresponding nucleic acid sequences may in some cases be madewithout a loss of function of the polypeptide or nucleic acid fragment.Substitutions may include, e.g., 1, 2, 3, or more residues. The aminoacid residues described herein employ either the single letter aminoacid designator or the three-letter abbreviation. Abbreviations usedherein are in keeping with the standard polypeptide nomenclature, J.Biol. Chem., (1969), 243, 3552-3559. All amino acid residue sequencesare represented herein by formulae with left and right orientation inthe conventional direction of amino-terminus to carboxy-terminus.

In some cases a determination of the percent identity of a peptide to asequence set forth herein may be required. In such cases, the percentidentity is measured in terms of the number of residues of the peptide,or a portion of the peptide. A polypeptide of, e.g., 90% identity, mayalso be a portion of a larger peptide Variations of the disclosedpolypeptide sequences include polypeptides or functional polypeptideshaving about 83% identity (e.g., 1 of 6 substituted) or about 67%identity (e.g., 2 of 6 substituted).

The term purified as used herein with reference to a polypeptide refersto a polypeptide that either has no naturally occurring counterpart(e.g., a peptidomimetic), or has been chemically synthesized and is thussubstantially uncontaminated by other polypeptides, or has beenseparated or purified from other most cellular components by which it isnaturally accompanied (e.g., other cellular proteins, polynucleotides,or cellular components). An example of a purified polypeptide is onethat is at least 70%, by dry weight, free from the proteins andnaturally occurring organic molecules with which it naturallyassociates. A preparation of the a purified polypeptide therefore canbe, for example, at least 80%, at least 90%, or at least 99%, by dryweight, the polypeptide. Polypeptides also can be engineered to containa tag sequence (e.g., a polyhistidine tag, a myc tag, or a Flag® tag)that facilitates the polypeptide to be purified or marked (e.g.,captured onto an affinity matrix, visualized under a microscope). Thus apurified composition that comprises a polypeptide refers to a purifiedpolypeptide unless otherwise indicated.

Polypeptides may include a chemical modification; a term that, in thiscontext, refers to a change in the naturally-occurring chemicalstructure of amino acids. Such modifications may be made to a side chainor a terminus, e.g., changing the amino-terminus or carboxyl terminus.In some embodiments, the modifications are useful for creating chemicalgroups that may conveniently be used to link the polypeptides to othermaterials, or to attach a therapeutic agent.

Nanoparticles

As demonstrated by the foregoing examples, the cartilage tissue-bindingligands may be used to target nanoparticles to a cartilage tissue, andsuch nanoparticles may include a therapeutic agent, for example, one ormore of the therapeutic agents described herein. While certainpolymer-therapeutics have been described elsewhere, these have mainlybeen designed to augment drug concentrations in tumor tissues¹. Suchalteration of the biodistribution of anticancer drugs through deliverysystems aims at reducing the drug's toxicity and at improvingtherapeutic effects^(2,3). To be effective, a drug delivery system mustescape non-specific systemic accumulation and phagocytotic clearance bythe host defense immune system. Moreover, after accumulation at thetarget site, penetration of the often avascular tissue must be achievedand the drugs released in active forms in order to exert the therapeuticeffect. For targeting articular cartilage, non-specific systemicaccumulation can be avoided by direct intra-articular injection. Whilethis is an attractive treatment approach because it minimizes systemiceffects, small compounds are prone to rapid lymphatic clearance andpossess a residence time of as short as 1-5 hours⁴.

In order to minimize intra-articular injections and to increase thebioavailability of drugs in articular cartilage, the nanoparticle-basedtherapeutic agent delivery system described herein exhibits activetargeting functionality for the cartilage, specifically, cartilageextra-cellular matrix. The combination of targeting functionality and ananoparticle-based delivery system enables better control ofbioavailability and biodistribution, particularly for intra-articulardrug delivery to the cartilage matrix. Surface functionalisation ofnanoparticles with the selected peptides controls the biodistribution byspecific accumulation of nanoparticles in the articular cartilage,specifically in the extra-cellular matrix. The cartilage matrix itselftherefore serves as a reservoir of nanoparticle encapsulated therapeuticmolecules, which are delivered to the site of the disease process.

As explained herein, articular cartilage can be targeted in vivo withnanoparticles by the use of polypeptides which have been characterizedto exhibit specific homing activity to articular cartilage. While PPSnanoparticles are used herein for demonstrative purposes, othertechniques for making nanoparticles may also be adapted. As exemplifiedwith the inverse emulsion polymerisation technique for PPS nanoparticlepreparation by which the emulsifier remains displayed on the surface¹⁴,the targeting polypeptides were made bioavailable by exposure at asurface of the nanoparticles. Size control was achieved in thisparticular technique by adjusting the emulsifier to monomer ratio andyielded sizes ranging from about 20 nm to about 200 nm¹⁴. While thedensity of the cartilage extra-cellular matrix represents a relevantobstacle not just for the screening of combinatorial peptide librariesbut potentially also for drug delivery to the cartilage, the use ofsuitably-sized nanoparticles and/or ligands with specific binding to thecartilage enhances delivery efficiency.

Nanoparticles are be prepared as collections of particles having anaverage diameter of between about 10 nm and about 200 nm, including allranges and values between the explicitly articulated bounds, e.g., fromabout 20 to about 200, and from about 20 to about 40, to about 70, or toabout 100 nm, depending on the polydispersity which is yielded by thepreparative method. Detailed methods for making and deliveringnanoparticles are set forth below and in U.S. Pat. Ser. No. 60/775,132,filed Feb. 21, 2006, which is hereby incorporated by reference herein.Numerous nanoparticle systems can be utilized, such as those formed fromcopolymers of poly(ethylene glycol) and poly(lactic acid), those formedfrom copolymers of poly(ethylene oxide) and poly(beta-amino ester), andthose formed from proteins such as serum albumin. Other nanoparticlesystems are known to those skilled in these arts. See also Devalapallyet al., Cancer Chemother Pharmacol., 07-25-06; Langer et al.,International Journal of Pharmaceutics, 257:169-180 (2003); and Tobio etal., Pharmaceutical Research, 15(2):270-275 (1998).

Larger particles of more than about 200 nm average diameterincorporating the cartilage tissue-binding ligands may also be prepared,with these particles being termed microparticles herein since they beginto approach the micron scale and fall approximately within the limit ofoptical resolution. For instance, certain techniques for makingmicroparticles are set forth in U.S. Pat. Nos. 5,227,165, 6,022,564,6,090,925, and 6,224,794.

Functionalization of nanoparticles to employ targeting capabilityrequires association of the targeting polypeptide with the particle,e.g., by covalent binding using a bioconjugation technique, with choiceof a particular technique being guided by the particle or nanoparticle,or other construct, that the polypeptide is to be joined to. In general,many bioconjugation techniques for attaching peptides to other materialsare well known and the most suitable technique may be chosen for aparticular material. For instance, additional amino acids may beattached to the polypeptide sequences, such as a cysteine in the case ofattaching the polypeptide to thiol-reactive molecules. Herein isdescribed an example of conjugation of the polypeptide WYRGRL (SEQ IDNO:1) with a cysteine at the C-terminus to Pluronic-F127 which wasderivatized with thiol-reactive vinyl sulfone. The polypeptide wascovalently bound to Pluronic by Michael-type addition and cansubsequently be used for nanoparticle synthesis.

Proteins Targeted to Articular Cartilage Tissue

Therapeutic agents such as therapeutic polypeptides can be furnishedwith targeting capability by the use of the polypeptide ligandsdescribed and advantageously exhibit longer retention times in a joint,for instance by making a fusion protein of a polypeptide ligand and atherapeutic protein. By designing gene specific primers for thetherapeutic polypeptide to be expressed, the polypeptide ligands can beattached to the N- or C-terminus in normal or reverse order. One of theprimers, forward or reverse depending whether the polypeptide ligand issupposed to be localized at the N- or C-terminus, contains a genesequence of the appropriate therapeutic polypeptide. For example, tomake a fusion protein of a given therapeutic polypeptide and (GGG)WYRGRL(SEQ ID NO:8) ligand at the C-terminus, the reverse primer correspondsto 5′-ctgatgcggccgctcTCACAGCCTGCCCCTATACCAGCCGCCGCCxxxxx-3′ (SEQ IDNO:9) which contains the codon sequence for WYRGRL in reverse complementwith a N-terminal glycine (GGG) linker (capital letters), as well as astop codon and a restriction site for NotI and an overhang. The Xscorrespond to the therapeutic polypeptide specific sequence and may be,e.g., around 20 base pairs long, although other lengths may be used asper conventional practice in these arts. The same example with samerestriction site for a N-terminal localization of WYRGRL(GGG) (SEQ IDNO:10) corresponds to5′-atcaggageggccgcTGGTATAGGGGCAGGCTGGGCGGCGGCxxxxx-3′ (SEQ ID NO:11) orconservative substitutions of the codon sequence. Instead of the threeglycines as a linker, other linkers appropriate to the properties of thetherapeutic polypeptides can be chosen. Similarly, other target ligandsmay be encoded, e.g., using thea nucleic acid sequence5′-ctgatgeggccgctcAAGATGGAAATGAGGATCGCCGCCGCCxxxxx-3′ (SEQ ID NO: 12)that endocdes DPHFHLGGG (SEQ ID NO:13) or the nucleic acid5′-ctgatgcggccgctcACGAACAAGCATAACACGGCCGCCGCCxxxxx-3′(SEQ ID NO:14) thatencodes RVMLVRGGG (SEQ ID NO:15). Thus a DNA sequence for WYRGRL (SEQ IDNO:1) is TGGTATAGGGGCAGGCTG (SEQ ID NO: 16), and for DPHFHL (SEQ IDNO:2) is AAGATGGAAATGAGGATC (SEQ ID NO:17), and for RVMLVR (SEQ ID NO:3)is ACGAACAAGCATAACACG (SEQ ID NO:18).

Certain therapeutic polypeptides include proteins present in cartilage,e.g., tissue inhibitors of matrix metalloproteinase-3 (TIMP-3), growthfactors (e.g., Transforming growth factor-beta (TGF-β), growthdevelopmental factor-5 (GDF-5), CYR61 (Cystein-rich61)/CTGF (connectivetissue growth factor)/NOV (Nephroblastoma overexpressed) (CCN2),insulin-like growth factor-1 (IGF-1), and bone morphogenic proteins(BMPs). Certain embodiments are molecules for viscosupplementation,e.g., molecules found in human cartilage that include chondroitinsulfate, keratane sulfate, hyaluronic acid, proteoglycans.

In some embodiments, a fusion protein is prepared and introduced intothe body as a purified composition in a pharmaceutically acceptablecondition, or with a pharmaceutical excipient. In certain embodiments,the fusion protein is produced using a cell, either a procaryotic or aeucaryotic cell. In other embodiments, nucleic acids encoding a fusionprotein are introduced into a patient, in which case the nucleic acidsmay be “naked” or part of a larger construct, e.g., a vector. In otherembodiments, transfected cells are introduced into a patient. The siteof introduction may be, e.g., systemic, in a joint, or in a cartilagetissue.

Cartilage Binding Fusion Protein: Targeted Recombinant TIMP-3

Tissue inhibitor of matrix metalloproteinase 3 (TIMP-3) is a relativelyinsoluble matricellular protein³⁰ which inhibits several matrixmetalloproteinases (MMP-1, -2, -9)³¹ in addition to aggrecanase 1 and 2,i.e. ADAMTS4 and ADAMTS5³². As such, this molecule is beneficial in thetreatment of osteoarthritis in any joint, specifically in a settingwhere the initiating mechanical cause is surgically corrected. BecauseTIMP-3 blocks these matrix degradative enzymes, the equilibrium inmatrix turn-over of articular cartilage may be restored by preventingfurther degradation. TIMP-3 is not specifically expressed in articularcartilage, however, and due to its ability of inhibiting several enzymesmay have severe potential adverse effects upon systemic administrationor systemic dissemination following intra-articular injection. Targeteddelivery of the inhibitory domain of TIMP-3 to articular cartilage canprevent potential systemic dissemination while increasing thetherapeutic effect of the molecule in the cartilage matrix, i.e. at thesite of the disease process in osteoarthritis. Moreover, the inhibitorydomain of TIMP-3 also serves as a protectant against cartilagedegradation in conditions such as rheumatoid arthritis, bacterialarthritis or reactive arthritis. Disclosed herein is a targetedrecombinant TIMP-3 (trTIMP-3) (fusion protein which contains one of thesequences (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3) or conservativesubstitutions thereof, e.g, at the N- or C-terminus. While TIMP-3 hasbeen selected as an embodiment, other protease inhibitors or matrixmetalloproteinase inhibitors may also be made and used by following thegeneral procedures disclosed herein.

TIMP-3 contains a signal domain, residues 1 to 24, the N-terminalinhibitory domain, residues 24 to 143, and an extra-cellular matrixbinding domain, residues 143 to 211 (SwissProt entry P35625). It hasbeen demonstrated that N-TIMP-3, i.e. residues 24 to 143, is sufficientfor exhibiting inhibitory activities on ADAMTS4 and ADAMTS5 as well asMMP-1 and MMP-2³. Accordingly, it is possible to engineer a fusionprotein of N-TIMP-3 (Cys24 to Asn 143) which contains the sequenceWYRGRL (SEQ ID NO:1) at the C-terminus. The natural non-specificC-terminal extra-cellular matrix binding domain is substituted with atargeting polypeptide specific for articular cartilage.

Human (P35625) and mouse N-TIMP-3 (P39876) share 97% sequence homologydiffering in three amino acid residues. Two of these residues representconservative substitutions, Thr vs. Ser at residue 74 and Asp vs. Glu atresidue 110. At residue 126 His substituted for Gln, which lies in adomain linking two α-chains.

For cloning, the sequence in NM_(—)000362 was used for primer design.The primers were designed as explained above (see SEQ ID NO:9 forreversed primer and SEQ ID NO:11 for the forward primer). RNA wasisolated using a RNeasy MinElute spin column (Qiagen, Hombrechikon,Switzerland) from immature murine articular chondrocytes which have beenisolated from epiphyseal cartilage of neonatal mice using a two stepdigestion protocol with collagenase D (Roche, Basel, Switzerland).First-strand cDNA was generated using SuperScript III (Invitrogen,Carlsbad, Calif.) with an Oligo-dT(20) primer (Microsynth, Balgach,Switzerland). RT-PCR for amplifying the required DNA fragments wascarried out with a proof-reading DNA polymerase (Pfu Turbo polymerase,Stratagene, LaJolla, Calif.) and the fragments with a required length of420 bp checked by agarose-gel electrophoresis. The DNA fragments weregel purified with a Nucleospin II column (Macherey-Nagel, Düren,Germany), cut with the corresponding restriction enzymes BamHI and NotI(New England BioLabs Inc., Ipswich, Mass.) and ligated into thebacterial protein expression vector pGEX-4T-1 (Amersham Biosciences, GEHealthcare Europe, Otelfingen, Switzerland), which expresses trTIMP-3 asa fusion protein with glutathione-S-transferase (GST) for purification,total MW 40 kDa. For bacterial expression, E. coli strain BL21 wastransformed with the trTIMP-3-pGEX construct by electroporation. Anoptimal expression clone was selected by anti-GST ELISA.

Bacterial cultures were grown in 2×YT until they reached an A₆₀₀ of 0.6to 0.7. Protein expression was then induced by 0.5M IPTG for 3 hours. Tocollect the protein from inclusion bodies, the bacteria werecentrifuged, lysed by sonication and centrifuged again. The pellet wasresuspended in a denaturing buffer (0.1M Tris-HCl, 50 mM glycine, 8 mMβ-mercaptoethanol, 8M urea, 0.2M PMSF, pH 8.0) and stirred overnight,centrifuged for 20 min. at 48000×g and the supernatant collected forrefolding. Refolding was performed by dialysing (24 kDa MWCO) thesupernatant against a large volume of refolding buffer at 4° C. withdecreasing amounts of urea to slowly remove the denaturing agent (0.1MTris-HCl, 1 mM EDTA, 0.2 mM PMSF, pH 8.0 supplemented with 4M, 2M, 1Mand 0M urea each for 24 hrs). The last step of dialysis was carried outagainst PBS for 24 hours. The refolded protein was freed fromprecipitations by centrifugation and subsequently purified by FPLC witha GST binding column (GSTrap FF, Amersham Biosciences, GE HealthcareEurope, Otelfingen, Switzerland). The GST tag of purified trTIMP-3-GSTfusion protein was cleaved of by incubation with thrombin for 24 hoursand purified by FPLC from GST with the GSTrap FF column and fromthrombin with a HiTrap Benzamidine FF column (Amersham Biosciences, GEHealthcare Europe, Otelfingen, Switzerland). Overall yield of trTIMP-3was about 1.5 mg/l of bacterial culture.

The activity of purified trTIMP-3 was assessed by MMP-2 zymography inwhich an equimolar amount of trTIMP-3 completely inhibits MMP-2 activityindicating a nearly 100% activity of the purified protein. Furtherexperiments could be used to demonstrate the aggrecanase inhibitingactivity of trTIMP-3 and the targeting specificity to articularcartilage in a similar fashion as shown for the nanoparticles describedherein. In addition, its therapeutic potential to prevent cartilagedegradation could be demonstrated in a suitable animal model, e.g., in amouse knee instability model.

Vectors

Accordingly, certain embodiments are directed to vectors for expressionof a therapeutic protein of interest, e.g., a therapeutic agent and apolypeptide ligand. Nucleic acids encoding a polypeptide can beincorporated into vectors. As used herein, a vector is a replicon, suchas a plasmid, phage, or cosmid, into which another nucleic acid segmentmay be inserted so as to bring about replication of the insertedsegment. Vectors of the invention typically are expression vectorscontaining an inserted nucleic acid segment that is operably linked toexpression control sequences. An expression vector is a vector thatincludes one or more expression control sequences, and an expressioncontrol sequence is a DNA sequence that controls and regulates thetranscription and/or translation of another DNA sequence. Expressioncontrol sequences include, for example, promoter sequences,transcriptional enhancer elements, and any other nucleic acid elementsrequired for RNA polymerase binding, initiation, or termination oftranscription. With respect to expression control sequences, “operablylinked” means that the expression control sequence and the insertednucleic acid sequence of interest are positioned such that the insertedsequence is transcribed (e.g., when the vector is introduced into a hostcell). For example, a DNA sequence is operably linked to anexpression-control sequence, such as a promoter when the expressioncontrol sequence controls and regulates the transcription andtranslation of that DNA sequence. The term “operably linked” includeshaving an appropriate start signal (e.g., ATG) in front of the DNAsequence to be expressed and maintaining the correct reading frame topermit expression of the DNA sequence under the control of theexpression control sequence to yield production of the desired proteinproduct. Examples of vectors include: plasmids, adenovirus,Adeno-Associated Virus (AAV), Lentivirus (FIV), Retrovirus (MoMLV), andtransposons. There are a variety of promoters that could be usedincluding, but not limited to, constitutive promoters, tissue-specificpromoters, inducible promoters, and the like. Promoters are regulatorysignals that bind RNA polymerase in a cell to initiate transcription ofa downstream (3′ direction) coding sequence.

Targeted Deliver of Viscosupplementation

Molecules for viscosupplementation may be conjugated with polypeptideligands described herein to form a conjugate for enhanced delivery andeffect in the cartilage. The formation of such conjugates is within theskill of ordinary artisans and various techniques are known foraccomplishing the conjugation, with the choice of the particulartechnique being guided by the materials to be conjugated. Suchconjugates may be delivered systemically or locally, e.g., orally or byinjection to a joint. Thus hyaluronic acid conjugation with apolypeptide ligand disclosed herein may prolong the retention time ofhyaluronic acid in the joint and therefore enhance the efficacy ofintra-articular viscosupplementation with hyaluronic acid. Conjugationof the polypeptide to hyaluronic acid can be performed either directlyas described above, or by the use of a polymer linker. Examples ofpolymer linkers are biocompatible hydrophilic polymers, includingpolymers free of amino-acids. For instance, a polymer linker may be apolyethylene glycol (PEG). Hyaluronic acid can be functionalized withacrylated PEG-Arg-Gly-Asp conjugates created by Michael-type additionchemistry (Park et al. Biomaterials 2003; 24:893-900). In general,polymers described herein for use with the polypeptide ligands may befree of amino acids, meaning that such polymers do not contain a naturalor synthetic amino acid.

In some embodiments, the conjugate is prepared and introduced into thebody as a purified composition in a pharmaceutically acceptablecondition, or with a pharmaceutical excipient. In certain embodiments,the conjugate is produced using a cell, either a procaryotic or aeucaryotic cell, as in the case of a biopolymer. In other embodiments,transfected cells are introduced into a patient. The site ofintroduction may be, e.g., systemic, in a joint, or in a cartilagetissue.

Targeting Genes Condensed with Polymers with the Polypeptides Attached

A small gene delivery system will have advantages with respect topenetrating the dense matrix of cartilage tissue. In some embodiments,therefore, the delivery system uses significantly condensed DNA, toenter the cartilage matrix and transfect non-dividing quiescentchondrocytes or other cells. In some embodiments, therefore, polypeptideligands are attached to polymers which contain a nuclear localisationsequence to form a conjugate and are used to condense DNA to overcomechallenges to gene transfection in chondrocytes embedded in thecartilage matrix. Some aspects of these techniques have been describedby in Trentin et al. PNAS 2006; 103:2506-11 and J Control Release 2005;102:263-75. Thus in certain embodiments the conjugate associated withnucleic acids encoding a therapeutic polypeptide is prepared andintroduced into the body as a purified composition in a pharmaceuticallyacceptable condition, or with a pharmaceutical excipient. In certainembodiments, such a conjugate is produced using a cell, either aprocaryotic or a eucaryotic cell, as in the case of a biopolymer. Inother embodiments, transfected cells are introduced into a patient. Thesite of introduction may be, e.g., systemic, in a joint, or in acartilage tissue.

Therapeutic Agents Associated with Ligands for Delivery to Cartilage

Polypeptides as described herein can be attached to other polymersthrough bioconjugation. The formation of such conjugates is within theskill of ordinary artisans and various techniques are known foraccomplishing the conjugation, with the choice of the particulartechnique being guided by the materials to be conjugated. The additionof amino acids to the polypeptide (C- or N-terminal) which containionizable side chains, i.e. aspartic acid, glutamic acid, lysine,arginine, cysteine, histidine, or tyrosine, and are not contained in theactive portion of the polypeptide sequence, serve in their unprotonatedstate as a potent nucleophile to engage in various bioconjugationreactions with reactive groups attached to polymers, i.e. homo- orhetero-bi-functional PEG (e.g., Lutolf and Hubbell, Biomacromolecules2003; 4:713-22, Hermanson. Bioconjugate Techniques. London. AcademicPress Ltd; 1996). An application where this may be useful is again fortargeted delivery of a therapeutic agent. In some embodiments, the agentis attached to a soluble polymer, and may be administer to a patient ina pharmaceutically acceptable form. Or a drug may be encapsulated inpolymerosomes or vesicles or covalently attached to polymers. In thelatter case, drugs are attached to the polymer backbone with adegradable site-specific spacer or linker (Lu et al. J Control Release2002; 78:165-73).

In general, soluble hydrophilic biocompatible polymers may be used toensure that the conjugate is soluble and will be bioavailable afterintroduction into the patient. Examples of soluble polymers arepolyvinyl alcohols, polyethylene imines, and polyethylene glycols (aterm including polyethylene oxides) having a molecular weight of atleast 100, 400, or between 100 and 400,000 (with all ranges and valuesbetween these explicit values being contemplated). Solubility refers toa solubility in water or physiological saline of at least 1 gram perliter. Domains of biodegradable polymers may also be used, e.g.,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polycaprolactones, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, and polycyanoacylates.

In some embodiments, a polypeptide-polymer association, e.g., aconjugate, is prepared and introduced into the body as a purifiedcomposition in a pharmaceutically acceptable condition, or with apharmaceutical excipient. The site of introduction may be, e.g.,systemic, in a joint, or in a cartilage tissue.

Cartilage Defect Treatment Using Polypeptide Ligands with SpecificBinding for Cartilage

The polypeptides ligands were discovered based on their binding affinityfor the cartilage matrix but are not specifically bound to the articularsurface. Therefore, besides embodiments such as targeting the cartilagematrix by therapeutic agent delivery systems or with engineered fusionproteins, the polypeptides are particularly well suited to target adefect because extracellular matrix is exposed at the defect. Thisfeature is useful for delivering biomaterials for gene, protein and/orcell delivery to mediate cartilage defect repair. Accordingly,embodiments include treating a defect in an articular cartilage usingone of the embodiments set forth herein. In fact, giving a biomaterialthe capability of adhering or otherwise specifically binding to acartilage defect does not only allow for injectable defect repairstrategies but also may enhance retention of the biomaterial in thecartilage defect. A variety of chemical schemes can be used toincorporate the cartilage-binding polypeptide into the biomaterial. Forexample, using a material as described by Sawhney et al., a chemicalapproach for incorporation as described by Hern et al. can be employed(Sawhney et al. Macromolecules 1993; 26:581-587 and Hern et al. J.Biomed. Mater. Res. 1998; 39:266-276). As another example, using amaterial as described by Lutolf et al., a chemical approach forincorporation as described therein can be employed (Lutolf et al. NatureBiotechnol. 2003; 21:513-518). In general, the modification of suchbiomaterials is within the skill of ordinary artisans and varioustechniques are known for accomplishing the modification, with the choiceof a particular technique being guided by the biomaterial and peptidesto be conjugated.

Specific binding, as that term is commonly used in the biological arts,generally refers to a molecule that binds to a target with a relativelyhigh affinity compared to non-target tissues, and generally involves aplurality of non-covalent interactions, such as electrostaticinteractions, van der Waals interactions, hydrogen bonding, and thelike. Specific binding interactions characterize antibody-antigenbinding, enzyme-substrate binding, and specifically bindingprotein-receptor interactions; while such molecules may bind tissuesbesides their targets from time to time, such binding is said to lackspecificity and is not specific binding. The peptides of SEQ ID NOs 1,2, and 3 may bind non-cartilage tissues in some circumstances but suchbinding has been observed to be non-specific, as evidenced by the muchgreater binding of the peptides to the targeted tissue as opposed tosurrounding joint tissues (data not shown).

Accordingly, embodiments include biomaterials comprising at least one ofthe ligands disclosed herein that are used to fill or augment a defectin a cartilage. A defect refers to a void in a surface (e.g., a pit,tear, or hole) or a pathological discontinuity in a surface (e.g., atear or eroded member). Fill refers to essentially filling or coveringthe defect or bridging the discontinuity. Augment refers to at leastpartial filling. In some embodiments, the biomaterial is a solid priorto placement in a patient, while in other embodiments the material ismade is situ, meaning it is formed from precursors at the site of thedefect. Thus biomaterials for cartilage defects may be supplemented witha ligand or other embodiment set forth herein. Examples of suchbiomaterials include U.S. Pat. Nos. 5,874,500, and 5,410,016, and whichinclude materials formed by in-situ polymerization. Biomaterials fortargeting cartilage defects, i.e. adhering to a cartilage defect, aresuited for the delivery of therapeutic agents to mediate cartilagerepair such as growth factors and for cell delivery to the repair site.In accordance with techniques for autologous cartilagetransplantation/implantation the use of a biomaterial for cell deliverywhich adheres to the defect by the use of polypeptide ligands (SEQ IDNO:1 through 3) and acts as a morphogenic guide may improve cartilagedefect repair.

Nucleic Acids

Certain embodiments are directed to nucleic acids. As used herein, theterm nucleic acid refers to both RNA and DNA, including siRNA, shRNA,miRNA, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA,as well as naturally occurring and chemically modified nucleic acids,e.g., synthetic bases or alternative backbones. A nucleic acid moleculecan be double-stranded or single-stranded (i.e., a sense or an antisensesingle strand). An isolated nucleic acid refers to a nucleic acid thatis separated from other nucleic acid bases that are present in a genome,including nucleic acids that normally flank one or both sides of anucleic acid sequence in a vertebrate genome (e.g., nucleic acids thatflank a gene). A conservatively substituted nucleic acid refers to thesubstitution of a nucleic acid codon with another codon that encodes thesame amino acid and also refers to nucleic acids that encodeconservatively substituted amino acids, as described herein with respectto polypeptides. Significantly, the combination of potential codons fora polypeptide of only about six residues is manageably small.

The nucleic acid sequences set forth herein are intended to representboth DNA and RNA sequences, according to the conventional practice ofallowing the abbreviation “T” stand for “T” or for “U”, as the case maybe, for DNA or RNA. Polynucleotides are nucleic acid molecules of atleast three nucleotide subunits. Polynucleotide analogues or polynucleicacids are chemically modified polynucleotides or polynucleic acids. Insome embodiments, polynucleotide analogues can be generated by replacingportions of the sugar-phosphate backbone of a polynucleotide withalternative functional groups. Morpholino-modified polynucleotides,referred to herein as “morpholinos,” are polynucleotide analogues inwhich the bases are linked by a morpholino-phosphorodiamidate backbone(see, e.g., U.S. Pat. Nos. 5,142,047 and 5,185,444). In addition tomorpholinos, other examples of polynucleotide analogues includeanalogues in which the bases are linked by a polyvinyl backbone, peptidenucleic acids (PNAs) in which the bases are linked by amide bonds formedby pseudopeptide 2-aminoethyl-glycine groups, analogues in which thenucleoside subunits are linked by methylphosphonate groups, analogues inwhich the phosphate residues linking nucleoside subunits are replaced byphosphoroamidate groups, and phosphorothioated DNAs, analoguescontaining sugar moieties that have 2′ O-methyl group). Polynucleotidesof the invention can be produced through the well-known and routinelyused technique of solid phase synthesis. Alternatively, other suitablemethods for such synthesis can be used (e.g., common molecular cloningand chemical nucleic acid synthesis techniques). Similar techniques alsocan be used to prepare polynucleotide analogues such as morpholinos orphosphorothioate derivatives. In addition, polynucleotides andpolynucleotide analogues can be obtained commercially. Foroligonucleotides, examples of pharmaceutically acceptable compositionsare salts that include, e.g., (a) salts formed with cations such assodium, potassium, ammonium, etc.; (b) acid addition salts formed withinorganic acids, for example, hydrochloric acid, hydrobromic acid (c)salts formed with organic acids e.g., for example, acetic acid, oxalicacid, tartaric acid; and (d) salts formed from elemental anions e.g.,chlorine, bromine, and iodine.

Pharmaceutical Carriers

Pharmaceutically acceptable carriers or excipient may be used to deliverembodiments as described herein. Excipient refers to an inert substanceused as a diluent or vehicle for a therapeutic agent. Pharmaceuticallyacceptable carriers are used, in general, with a compound so as to makethe compound useful for a therapy or as a product. In general, for anysubstance, a pharmaceutically acceptable carrier is a material that iscombined with the substance for delivery to an animal. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. In some cases the carrier isessential for delivery, e.g., to solubilize an insoluble compound forliquid delivery; a buffer for control of the pH of the substance topreserve its activity; or a diluent to prevent loss of the substance inthe storage vessel. In other cases, however, the carrier is forconvenience, e.g., a liquid for more convenient administration.Pharmaceutically acceptable salts of the compounds described herein maybe synthesized according to methods known to those skilled in this arts.Thus a pharmaceutically acceptable composition has a carrier, salt, orexcipient suited to administration to a patient. Moreover, inertcomponents of such compositions are biocompatible and not toxic.

The compounds described herein are typically to be administered inadmixture with suitable pharmaceutical diluents, excipients, extenders,or carriers (termed herein as a pharmaceutically acceptable carrier, ora carrier) suitably selected with respect to the intended form ofadministration and as consistent with conventional pharmaceuticalpractices. Thus the deliverable compound may be made in a form suitablefor oral, rectal, topical, intravenous injection, intra-articularinjection, or parenteral administration. Carriers include solids orliquids, and the type of carrier is chosen based on the type ofadministration being used. Suitable binders, lubricants, disintegratingagents, coloring agents, flavoring agents, flow-inducing agents, andmelting agents may be included as carriers, e.g., for pills. Forinstance, an active component can be combined with an oral, non-toxic,pharmaceutically acceptable, inert carrier such as lactose, gelatin,agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.The compounds can be administered orally in solid dosage forms, such ascapsules, tablets, and powders, or in liquid dosage forms, such aselixirs, syrups, and suspensions. The active compounds can also beadministered parentally, in sterile liquid dosage forms. Buffers forachieving a physiological pH or osmolarity may also be used.

All patent applications, patents, and publications mentioned herein arehereby incorporated by reference herein to the extent they do notdirectly contradict the explicit disclosures set forth herein.

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This application discloses various inventive embodiments that each havecertain features. In general, these features may be mixed-and-matchedwith each other to create additional functional embodiments.

1. A substantially pure polypeptide not found in nature comprising anamino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or aconservative substitution thereof, wherein the polypeptide specificallybinds a cartilage tissue.
 2. The polypeptide of claim 1 wherein thesequence length is between 6 and 1000 residues.
 3. The polypeptide ofclaim 1 comprising a therapeutic agent polypeptide.
 4. The polypeptideof claim 3 wherein the therapeutic agent polypeptide is tissue inhibitorof matrix metalloproteinases-3 (TIMP-3).
 5. The polypeptide of claim 1comprising a synthetic backbone linkage.
 6. A substantially pure nucleicacid not found in nature that comprises a nucleic acid sequence thatencodes SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a conservativesubstitution thereof, wherein a polypeptide encoded by the nucleic acidspecifically binds to a cartilage tissue.
 7. The nucleic acid of claim 6wherein the polypeptide that is encoded comprises a therapeutic agentpolypeptide.
 8. A vector comprising the nucleic acid of claim
 6. 9. Amethod of treating cartilage of a mammal comprising administering to themammal a pharmaceutically acceptable composition that comprises thepolypeptide of claim 1, wherein the polypeptide targets the therapeuticagent to the cartilage tissue by specifically binding cartilage tissueof the mammal.
 10. A delivery system for delivering a therapeutic agentcomprising: a substantially purified preparation that comprises apharmaceutically acceptable excipient, a therapeutic agent, and apolypeptide ligand not found in nature comprising an amino acid sequencein the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, andconservative substitutions thereof, wherein the polypeptide ligandspecifically binds a cartilage tissue for targeted delivery of thetherapeutic agent to cartilage tissues.
 11. The delivery system of claim10 wherein the therapeutic agent comprises a drug, a visualizationagent, or a therapeutic polypeptide.
 12. The delivery system of claim 10comprising a fusion polypeptide that comprises the polypeptide ligandand the therapeutic agent.
 13. The delivery system of claim 10comprising a molecule that comprises covalent bonds to the polypeptideligand and the therapeutic agent.
 14. The delivery system of claim 10wherein the polypeptide ligand is covalently bonded to a biocompatiblepolymer that is associated with the therapeutic agent.
 15. The deliverysystem of claim 14 wherein the biocompatible polymer is free of aminoacids.
 16. The delivery system of claim 10 comprising a collection ofnanoparticles having an average diameter of between about 10 nm andabout 200 nm, wherein the nanoparticles comprise the therapeutic agentand the polypeptide ligand.
 17. A method of treating cartilage of amammal comprising administering to the mammal a pharmaceuticallyacceptable composition that comprises the delivery system of claim 13,wherein the polypeptide targets the therapeutic agent to the cartilagetissue by specifically binding cartilage tissue of the mammal.
 18. Themethod of claim 17 wherein the composition is administeredintra-articularly.